Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – V. Reinterpretation of the experiments on in vivo action dichroism of phytochrome

Experiments by several authors on the effects of polarized light on phytochromemediated responses in fern gametophytes and in the green alga Mougeotia have earlier been interpreted as showing that the transition moment of phytochrome in the P_r form is parallel to the plasmalemma, but perpendicular to the plasmalemma for the P_fr form of phytochrome. It is now shown that the experimental results can be interpreted differently, and that they are also consistent with a chromophore rotation of about 30° (instead of 90°), as found for immobilized phytochrome molecules in vitro. Thus there is no evidence for a rotation of the whole phytochrome protein. For the gametophyte of Adiantum it is calculated that the P_r transition moment is inclined 17° to the plasmalemma, and the P_fr transition moment ca 50°, corresponding to an in vivo chromophore rotation of ca 33°; however, these values are very approximate.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – VI. Relation between the two pigments of the mycochrome system in Alternaria cichorii

Conidiation in Alternaria cichorii Nattras is reversibly stimulated by near ultraviolet radiation (NUV, ca 313 nm) and inhibited by blue light (ca 450 nm) and seems to be a mycochrome-mediated process. After induction with plane-polarized NUV, blue light polarized perpendicularly to the NUV was more effective in counteracting the induction than was blue light polarized parallel to the NUV. From this the conclusions are drawn that (a) both the blue-absorbing component (presumably a flavo-protein) and the P_NUV of the mycochrome system are membrane-bound and that (b) the transition moment associated with blue light absorption in the presumed flavoprotein forms an angle of at least 53° with the transition moment associated with NUV absorption in P_NUV.     

Synthesis of mannose oligosaccharides via reversal of the α-mannosidase reaction

Summary Three naturally occurring isomers of the disaccharideO--d-mannosyl-d-mannoside were synthesized by reversing the hydrolytic activity of jack bean -mannosidase at 75°C in a very high concentration of mannose. Higher oligosaccharides were also obtained at the later stages of the reaction. The maximum total yield of disaccharides was 37% (w/w) based on the total amount of saccharides.     

Spore production ofPenicillium roqueforti in fermentors filled with buckwheat seeds: batch and semi-continuous cultivation

Summary The behaviour of a drum fermentor and a column fermentor during the sporulation ofPenicillium roqueforti on buckwheat seeds is presented. The main problem encountered during the course of a cultivation is the free water released (about 0.1 ml/g dry matter) which must be removed from the medium. The rotation of the drum fermentor may disturb the growth and the sporulation. The column fermentor thus represents the best way to perform batch cultivation of the fungus: 10⁹ external spores/g dry matter are obtained.Semi-continous cultivation, with sequential emptying and filling, is performed in 1-liter bottles. This kind of cultivation may give a maximal average productivity close to 9.2·10⁶ external spores/g dry matter per hour. A drum fermentor, rotading only when emptying and filling, could represent an alternative to perform this kind of cultivation.     

Distribution of intrapleural and intravenous Corynebacterium parvum in humans; 99mTc−, and 131I-labeled bacteria

Summary The distribution of Corynebacterium parvum labeled with ¹³¹iodine or ^99mtechnetium was studied in 17 patients with bronchogenic carcinoma. The labeled bacteria were given intravenously or intrapleurally and monitored by whole-body gamma tracking and samples of blood and urine. Even though the rate of physical decay is quite different for ¹³¹iodine and ^99mtechnetium, the tracking time of labeled bacteria was limited to 24 h after injection for both radioactive isotopes. Technetium labeling was preferred because of greater imaging resolution and less radiation dose to the patient. Following intravenous administration, labeled C. parvum was found predominantly in the liver and spleen, and in a lesser amount in the lung. Radioactivity was confined to the pleural cavity after intrapleural injection. These results suggest the combined intravenous and intrapleural route of adjuvant immunosupportive agents such as C. parvum for operable lung cancer patients.     

Close linkage between X-linked ectodermal dysplasia and a cloned DNA sequence detecting a two allele restriction fragment length polymorphism in the region Xp11-q12

Summary EDA (ectodermal dysplasia, anhidrotic) is an X-linked recessive disorder characterized by hypohidrosis, hypoor anodontia, and hypotrichosis. A possible linkage between the gene for EDA and a number of restriction fragment length polymorphisms (RFLPs) spread over the X chromosome was investigated in two Danish families segregating EDA. No recombination between the gene for EDA and our probe pTAK8, which detects a two allele polymorphism in the region Xp11-q12, was found in nine informative meiotic events (seven of which are phase known), giving a maximal lod score of 2.41 at a recombination fraction of 0.00. This juxtacentromeric location of the gene for EDA agrees well with the linkage data obtained with the other markers used in this study.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Application of Response Surface Methodology to Evaluation of Bioconversion Experimental Conditions

Using Candida tenuis, a yeast isolated from the digestive tube of the larva of Phoracantha semipunctata (Cerambycidae, Coleoptera), we were able to demonstrate the bioconversion of citronellal to citronellol. Response surface methodology was used to achieve the optimization of the experimental conditions for that bioconversion process. To study the proposed second-order polynomial model, we used a central composite experimental design with multiple linear regression to estimate the model coefficients of the five selected factors believed to influence the bioconversion process. Only four were demonstrated to be predominant: the incubation pH, temperature, time, and the amount of substrate. The best reduction yields (close to 90%) were obtained with alkaline pH conditions (pH 7.5), a low temperature (25°C), a small amount of substrate (15 μl), and short incubation time (16 h). This methodology was very efficient: only 36 experiments were necessary to assess these conditions, and model adequacy was very satisfactory as the coefficient of determination was 0.9411.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.